At 3 months, general symptoms associated with CMA showed significant improvement with the TAAF such as frequency of crying, irritability, and sleeping time and quality, and, with the RAAF only quality of daytime sleep (Table 1, Fig. 2, Table S2 [http://links.lww.com/MPG/A459]). In both the groups, daily crying time significantly decreased (Table 1). Significant differences between the 2 groups were observed for frequency of irritability and quality of nighttime sleep, with the TAAF being more effective (respectively P < 0.001 and P = 0.036, χ2 test and Fisher test).
At 3 months, infants’ growth was similar between the 2 groups with no significant differences for weight, length, weight-for-length, BMI, and head circumference z scores. Compared with baseline, growth during 6 months showed significant improvement of weight-for-age z score in the group initially fed the TAAF (mean ± SD 0.3 ± 0.6, Fig. 3). In the same group, length, weight-for-length, BMI, and head circumference z scores increased by 0.1 (±0.8), 0.1 (±0.8), 0.4 (±0.9), and 0.3 (±0.8), respectively, during the 6-month study. As infants of the RAAF group switched to TAAF during the last 3 months, mean changes of anthropometric data between 3 and 6 months were adjusted with baseline values as covariate. Based on these analyses, at 6 months, no significant differences were noted between groups. Between 3 and 6 months, the mean weight-for-age z score significantly increased by 0.1 ± 0.3 in the RAAF group (P = 0.028, Wilcoxon test).
The most common adverse events were gastrointestinal tract affections and infections and were not related to the study product. Incidence of adverse events was not different between groups. A total of 4 serious adverse effects were recorded between 1 and 3 months: 3 in the TAAF group (gastroenteritis, pneumonia, and gastrooesophageal reflux) and 1 in the RAAF group (gastroenteritis); 3 were recorded between 3 and 6 months (malaise, gastroenteritis, and pneumonia). None were related to the study formula and none led to study drop out. In 2014, Dupont et al (15) reported 2 nonserious adverse events that led to study termination within the first month in the RAAF group. Parents’ satisfaction with the allocated formula was high in both the groups (90.9% vs 79.0% at 1 month and 90.0% vs 91.7% at 3 months for TAAF and RAAF groups, respectively). Infants’ acceptability of the allocated formula was judged as very good or good by more parents in the TAAF group than in the RAAF group at 1 and 3 months (76.5% vs 58.6% at 1 month and 91.4% vs 61.9% at 3 months, not significant CMH test). No differences between groups were noted concerning the presence of gas and intestinal bloating at 1 and 3 months.
From inclusion to 3 months, there was a significant fall in mean plasma eosinophils concentrations in both the groups: from 0.49 (±0.55) to 0.28 (±0.22) 109/L in the TAAF group (n = 32, P = 0.017) and from 0.53 (±0.47) to 0.22 (±0.19) 109/L in the RAAF group (n = 29, P < 0.001, Wilcoxon test), with no significant difference between the groups. All of the other mean biochemical parameters, IgG, IgA, IgM, serum ferritin, and complete blood count were within normal range values at 3 months. In the subset of infants (25 in the TAAF group and 22 in the RAAF group) with plasma amino acid evaluation at 3 months, there were no differences between both the groups in plasma essential amino acid concentrations except for valine, higher in the RAAF group (P = 0.049, Wilcoxon test) (Fig. S1, http://links.lww.com/MPG/A460).
Faecal Analysis Data
At 3 months, infants of both the groups were colonized at high levels by bacterial groups usually encountered in the dominant microbiota of infants, that is, Bacteroides/Prevotella, C coccoides group, and Bifidobacterium (median >109 CFU/g of faeces, Fig. S2 [http://links.lww.com/MPG/A461]). A total of 2 infants, however, in the RAAF group were not colonized by bifidobacteria. Concerning the subdominant microbiota (median levels comprised between 105.5 and 109 CFU/g of faeces), >90% of the infants were colonized by E coli, Enterococcus, and Clostridium cluster I/II and cluster XI. By contrast, colonization occurred less frequently with the C leptum group, Lactobacillus/Leuconostoc group, and Staphylococcus (53% to 83% of the infants depending on the bacterial groups and the formula group). After 3 months of AAF feeding, the evolution in the total bacteria counts was significantly different between TAAF and RAAF groups (P = 0.021), with a stable bacterial count in the TAAF group and a slight increase in the RAAF group. Despite no significant differences between groups for any genera, some different trends in the evolution were observed. Bifidobacteria decreased in both the groups, but the decrease was moderate in the TAAF group, −0.21 log10 CFU/g (±0.45), and higher in the RAAF group, −1.15 log10 CFU/g (±0.52) (adjusted means). When expressed as percentage of total bacteria, this trend was more marked, with bifidobacteria remaining stable in the TAAF group, 1.7% (±8.7), and decreasing in the RAAF group, −20.3% (±10.1). Similar trend was observed for the Lactobacillus/Leuconostoc group: −0.43 log10 CFU/g (±0.51) in the TAAF group versus −1.01 log10 CFU/g (±0.59) in the RAAF group. Likewise, there were increases in percentages of Bacteroides/Prevotella and C coccoides groups in the TAAF group (Fig. S3, http://links.lww.com/MPG/A462). These modifications tended to modify the balance of the microbiota, with a trend to a higher abundance of the bifidobacteria, Bacteroides/Prevotella, and C coccoides groups in the TAAF group compared with the RAAF group at 3 months.
EDN values ranged from <120 to 3475 ng/g at inclusion and from <120 to 3324 ng/g at 3 months, showing a high interindividual variability (n = 38). The trend (median, range) was similar with a decrease for both the groups: −196 ng/g (−1954 to 2026) in the TAAF group and −166 ng/g (−2469 to 2832) in the RAAF group, with no significant difference between the groups.
This study demonstrates the efficacy and safety in the long term of both AAFs in infants with proven CMA and intolerance to eHFs. All of the infants tolerated their allocated AAF for 3 months, and the TAAF was also tolerated by all of the infants who completed the 6-month study, including the infants who switched from RAAF to TAAF at 3 months. As reported in 2014, at 1 month, a complete resolution of the major CMA symptom occurred in 61.9% and 51.5% of infants in TAAF and RAAF groups, respectively (15). Results presented here confirm that at 3 months, both AAF formulas improved the major CMA symptom, the percentage of resolution being significantly higher with the TAAF.
One could argue that a DBPCFC, considered as the criterion standard (3) for CMA diagnosis, was not performed in all of the patients. Only a minority of subjects (26.7%) had no challenge and diagnosis based on (s)IgE assay and SPT values above validated cutoff levels for active CMA (19). This disposition in the protocol was chosen to favour the enrolment process of families dealing with an already complicated medical history (previous failure with 1 or more eHFs). In fact, the primary endpoint of this study, the tolerance/hypoallergenicity of the TAAF at 1 month in >90% (with 95% CI) of infants with both CMA and intolerance to eHFs, requires a sample size of ≥29 subjects with no reaction (2). This minimum number of subjects was reached in the TAAF group, even in the subgroup of subjects with CMA proven by a DBPCFC (15), and 100% of them tolerated the TAAF for ≥3 months. In addition, the percentage of the major CMA symptom resolution did not statistically differ between the infants with CMA proven by (s)IgE/SPT and those with CMA proven by a DBPCFC at 1 and 3 months; in the latter, this percentage remained significantly higher in the TAAF group compared with the RAAF group at 3 months.
In case of CMA, recommendations are first dietary treatment with eHFs to eliminate cow's-milk protein in the diet (1,2,4). eHFs have been successfully used to treat most of the infants with cow's-milk allergy. Some infants, however, are sensitive to these formulas, so their CMA symptoms persist with eHFs. AAFs are the recommended choice for these infants. Several studies reported hypoallergenicity and tolerance of AAF in infants with proven CMA (8,14,29) or in infants intolerant to eHFs (6,10), but no randomised controlled trial had ever been carried out in infants with CMA and allergy symptoms persisting with eHFs (3).
Beyond testing the tolerance of the AAFs, the aim was to quantify their efficacy in a large cohort of infants with both CMA and intolerance to eHFs. As CMA is characterized by a multitude of symptoms, including fussiness, irritability, emesis, poor feeding, and diarrhoea at presentation (1,4,30), paediatricians had to determine in this study the dominant allergic symptom for each subject at inclusion and to assess its evolution at each follow-up visit. This dominant allergic symptom, however, did not reflect the complete clinical situation of each patient: for the subjects in which this symptom was not improved after 1 month, other allergic symptoms were resolved or improved. In addition, in 1995, Hill et al (31) evaluated children with multiple food intolerance, including CMA, and requiring an AAF feeding. Their allergic symptoms were scored during an eHFs challenge and compared with an AAF, as placebo; the score was most of the time lower with the AAF than with the eHF but never equal to zero. Recently, another tool, the Cow's Milk-related Symptom Score (CoMiSS), was proposed to allow the assessment and quantification of the evolution of CMA symptoms during a therapeutic intervention (32). Several trials, in which children with CMA on an eviction diet were followed, reported the evolution of CoMiSS values; after 1 month of dietary treatment, these values significantly decreased but were not equal to zero (33–35). Finally, the percentages of resolution of the dominant allergic symptom did not differ at 1 month between the TAAF and the RAAF, the latter having been considered for decades as the reference for severe CMA treatment (6,7).
The wide array of symptoms studied allowed a large documentation of symptom evolution. Regarding cutaneous symptoms, mean SCORAD index values observed in infants with CMA at baseline ranged from 19.4 ± 16.1 (29) to 21 (95% CI 16–26) (5). Niggemann et al (13) reported a median SCORAD index of 18.5. All of these trials showed decreases of SCORAD index with AAFs. The present results confirm the efficacy of AAFs to reduce eczema severity in infants with CMA and intolerance to eHFs, the TAAF reducing significantly more the SCORAD index than the RAAF. The major difference between both AAFs and that may explain the observed difference is the presence of a pectin-based thickener in the TAAF. To the best of our knowledge, no clinical study has reported a significant effect of a pectin-based complex on skin health improvement in allergic infants. An earlier study found that a prebiotic mix, containing in particular acidic pectin oligosaccharides, had an effect on eczema prevention in infants with no atopy risk but no impact on eczema severity (36). In addition, results on the effect of prebiotic and probiotic blends on SCORAD evolution in infants and young children with atopic dermatitis are inconsistent (37,38). Given these contradictory results, the possible role of pectin on skin symptoms is unclear, requiring further investigation.
Previous studies suggest that pectin could play an integral role in improving stool consistency (34,35), and this was shown by a significant decrease in the number of liquid and hard stools in the TAAF group. Pectin is a dietary fibre known for having an impact on faecal weight (39). Other mechanisms could be considered such as enhanced colonic fluid absorption through the production of short-chain fatty acids (40) by colon microbiota (41), which may explain the positive effects of a pectin-based diet on diarrhoea resolution (42).
Very little data are available on the effect of AAFs on general symptoms related to CMA and affecting daily family life. Vanderhoof et al (7) reported the evolution of crying time and duration of sleep patterns in such patients. Fifteen days after AAF initiation, crying time was significantly reduced, but no change in the sleep duration was observed. The present results confirm the impact of AAF on reduced crying time but also show a significant increase in daily sleeping time. In addition, the TAAF showed a better improvement of irritability signs and quality of night sleep, compared with the RAAF.
Compared with healthy infants, allergic infants may have impaired growth, which is partially linked to improper food substitutions following allergen elimination (43). Moreover, CMA may also increase energy requirements because of inflammation (ie, skin or gastrointestinal) and disrupted sleep, and reduce the absorption of major nutrients (ie, CMA-induced enteropathy) (44). Mean weight and length z scores of the infants included in this study were <−0.5 at inclusion, showing poor weight and length gains in these infants, as already evidenced by other clinical studies (5,10,34,35,45,46). Previous clinical studies assessed the impact of AAF on the growth of healthy infants (8,9), or infants with CMA (5,13,29), but only 2 open noncontrolled studies reported growth data in infants with CMA and intolerance to eHFs (6,10). They all showed improvement of anthropometric data with AAF. In the present study, growth of infants fed the TAAF was similar to that of infants fed the RAAF, confirming the nutritional safety of this formula.
Essential amino acid plasma concentrations did not differ between the groups, except for valine, which was closer to breast-fed concentrations in the TAAF group, and slightly higher in the RAAF group compared with the TAAF group, however not clinically significant. They were all in the range of the concentrations measured in 6-month healthy infants (47) and similar to those in breast-fed infants (48).
Three months after AAF initiation, plasma eosinophils significantly decreased in both the groups. Same results were reported with AAF feeding but in older children (11,49). EDN is a faecal marker of intestinal immune stimulation related to allergic inflammatory responses, especially eosinophilic infiltration (18). Kalach et al (18) showed that infants with intestinal symptoms during a cow's milk challenge had higher faecal EDN levels than those with other symptoms or those tolerating cow's milk, suggesting an eosinophilic degranulation in the intestine. Our study confirms the high level of faecal EDN in this population. Three months after AAF initiation, children from both the groups showed a decrease of their EDN values, which may reflect a decrease of their intestinal inflammation.
Microbiota composition is in both groups in accordance with previous studies performed in infants at a similar age (50). The intervariability observed between infants is likely because of the diversity in age at sampling and in the perinatal determinants of the studied infants which are known to affect the bacterial colonization (50). No significant differences were observed between both the formulas throughout the study. Although the number of samples may not have been sufficient to detect a statistical difference, in the TAAF group, however, bifidobacteria percentages remained stable throughout the study whereas a slight decrease was observed in the RAAF group. Such observation was reported by Thompson-Chagoyan et al (51) who showed in CMA infants followed for 6 months on an elimination diet a significant increase in percentages in lactobacilli and a significant decrease in bifidobacteria. In TAAF, pectin, which is known to increase bifidobacteria counts (41), may have counterbalanced the effect of an elimination diet on decreasing bifidobacteria percentages.
This study presents several limitations. First, the number of faecal samples collected was low compared with the number of subjects included and may explain the absence of statistical differences observed between the groups. Secondly, our population may not have been homogenous enough regarding the CMA symptomatology to detect a possible impact of the dietary treatment on EDN values. In fact, this marker being associated with eosinophils intestinal infiltration, it may be more relevant in patients with gastrointestinal symptoms (18).
In conclusion, we have shown that the TAAF is hypoallergenic, efficient to alleviate symptoms, nutritionally adequate, and able to support growth during long-term feeding in these infants.
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